Tagging methods as a tool to investigate histone H3 methylation dynamics in mouse embryonic stem cells PhD Thesis Giovanni Ciotta Reviewers: Date of submission: 23.03.2011 Prof. Francis Stewart Date of defense: 20.05.2011 Prof. Constanze Bonifer INTERNATIONAL MAX PLANCK RESEARCH SCHOOL MAX PLANCK INSTITUTE OF MOLECULAR CELL BIOLOGY AND GENETICS Abstract Covalent modification of histones is an important factor in the regulation of the chromatin structure implicated in DNA replication, repair, recombination, and transcription, as well as in RNA processing. In recent years, histone methylation has emerged as one of the key modifications regulating chromatin function. However, the mechanisms involved are complex and not well understood. Histone 3 lysine 4 (H3K4) methylation is deposited by a family of histone H3K4 methyltransferases (HMTs) that share a conserved SET domain. In mammalian cells, six family members have been characterized: Setd1a and Setd1b (the mammalian orthologs of yeast Set1) and four Mixed lineage leukemia (Mll) family HMTs, which share limited similarity with yeast Set1 beyond the SET domain. Several studies demonstrated that the H3K4 methyltransferases exist as multiprotein complexes. To functionally dissect H3K4 methyltransferase complexes, GFP tagging of the core subunit Ash2l and the complex-specific subunits Cxxc1 and Wdr82 (Setd1a/b complexes) Men1 (Mll1/2 complexes), and Ptip (Mll3/Mll4 complexes), was used. The fusion proteins were successfully expressed in mouse embryonic stem cells (ES cells), analyzed by confocal microscopy, Mass Spectrometry (MS) and ChIP-seq. Ptip was the only subunit able to bind mitotic chromatin. Additionally, both Ptip and Wdr82 were found to associate with cell cycle regulators, suggesting a possible role of the two proteins or respective complexes in cell cycle regulation. Mass Spectrometry revealed that Wdr82 and Ptip interact with members of the PAF complex, and ChIP-seq showed that Wdr82, Cxxc1 and Ptip positively modulate pluripotency genes. Thus, Setd1a/b and Mll3/4 complexes might act together in the regulation of embryonic stem cells identity. Protein pull downs identified at least one new Setd1a/b interactor, Bod1l that is orthologous to the yeast protein Sgh1, a component of the Set1C complex. Furthermore, our MS and ChIP-seq data suggested that only Mll2 complex binds to bivalent promoters, wheras Mll2 and Setd1a complexes might function together in a set of promoters. Content 1. Introduction 1 1.1. Epigenetics 1 1.2. Chromatin structure 1 1.3. Chromatin remodeling 3 1.4. Post-translational histone modifications 4 1.5. Programming of gene expression by Polycomb (PcG) proteins and 8 Tritorax group (TrxG) proteins 1.6. The mammalian trx-related proteins 10 1.7. Shared and unique H3K4 methyltransferase complex components 12 1.8. Embryonic stem cells, protein tagging and recombineering as a 15 platform for functional genomics and proteomics 2. Aim of this work 21 3. Materials and Methods 23 3.1. Materials 23 3.1.1. General Materials 23 3.1.2. Chemical and Reagents 24 3.1.3. Cell culture media and supplements 24 3.1.4. Kits 24 3.1.5. Restriction endonucleases 25 3.1.6. Other reagents (markers, nucleotides, etc) 25 3.1.7. Antibodies 25 3.1.8. Plasmids 26 3.1.9. Synthetic oligonucleotides 26 3.1.10. PCR products 28 3.2. Methods 30 3.2.1. Obtaining a BAC 30 3.2.2. Design of the tagging cassette 31 3.2.3. Preparation of the tagging cassette by PCR 34 3.2.4. Transformation with Red/ET expression plasmid 35 3.2.5. Inserting the tagging cassette into a BAC 36 3.2.6. Quality control of recombineering 37 3.2.7. BAC/plasmid Mini-prep protocol and restriction analysis of DNA 38 3.2.8. BAC Maxi-prep Protocol 39 3.2.9. Generation of gene targeting constructs by recombineering 39 3.2.10. Subcloning of a specific region from a BAC by Red/ET 40 3.2.11. Verification and preparation of DNA for targeting 41 3.2.12. Culture conditions for mouse embryonic stem cells 42 3.2.13. Transfection of mouse ES cells to establish stable BAC transgenic lines 42 3.2.14. Electroporation of mouse ES cells 43 3.2.15. Picking colonies for clonal analysis 44 3.2.16. Genomic DNA extraction from 96 well 44 3.2.17. Long Range PCR analysis of targeted clones 45 3.2.18. Preparation of cells for Western blot analysis 46 3.2.19. Fluorescence analysis of the GFP expressing cell lines 47 3.2.20. Small scale immunoprecipitation of GFP/Venus-tagged 48 proteins 3.2.21. Immunoprecipitation of GFP-tagged proteins for mass 49 spectrometry 3.2.22. Chromatin Immunoprecipitation protocol for deep sequencing 51 analysis 3.2.23. qPCR analysis for the enrichment of the ChIP DNA on 55 specific loci 3.2.24. Deep sequencing analysis of the immunoprecipitated DNA 55 4. Results 57 4.1. Generation of C-terminal GFP tagged H3K4 methyltransferases complex- specific subunits 57 4.2. Generation of targeting vectors by subcloning 60 4.3. Generation of BAC transgenic mouse ES cell lines 61 4.4. Targeting of mouse ES cells by homologous recombination 62 4.5. Expression of the tagged proteins in mouse ES cell lines 63 4.6. Localization of the GFP tagged histone H3K4 66 methyltransferase complex-specific subunits 4.7. Protein immunoprecipitation of the tagged subunits 70 4.8. Mass Spectrometry analysis of the H3K4 methyltransferases 71 complex subunits 4.9. Bod1l and Dppa5a protein analysis 76 4.10. Tagging of the new interactors Bod1l and Dppa5a 78 4.11. Bod1l and Dppa5a expression in mouse ESCs 79 4.12. Bod1l and Dppa5a cellular localization in mouse ES cells 79 4.13. Bod1l and Dppa5a protein immunoprecipitation 80 4.14. ChIP assay of the H3K4 methyltransferase complexes 81 4.14.1. Genome-wide ChIP-seq analysis 83 4.14.2. Coverage of the tagged subunits at the H3K4 methyltransferase 86 promoters 4.14.3. Role of the H3K4 methyltransferases in the regulation of 87 pluripotency genes 4.14.4. Regulation of Bod1l gene expression by H3K4 methylation 89 4.14.5. Outlook of bivalent promoters 91 5. Discussion 94 5.1. Generation, expression and functional tests of the H3K4 94 methyltransferase complex-specific tagged components 5.2. Cellular localization of the GFP/YFP tagged proteins and their 97 regulation of the cell cycle 5.3. The novel components of H3K4 methyltransferases 100 complexes 5.4. Regulation of promoters and pluripotency by H3K4 102 methylation 5.5. Insights into bivalency of promoters 106 5.6. Model for regulation of chromatin state in pluripotent and 107 lineage-committed cells 6. Summary 109 7. Abbreviations 111 8. References 113 Aknowledgements 133 Statement of originality 135 1. Introduction 1.1. Epigenetics Genomic DNA is the ultimate template of our heredity. Despite the excitement over the human genome, many challenges remain in understanding the regulation and transduction of genetic information. DNA alone does not provide the full range of information that finally results in a complex eukaryotic organism. All somatic cells obviously contain the same genetic information, but each cell type has a specific gene expression pattern. There are several layers of regulation of gene expression. One way that genes are regulated is through the remodeling and modification of chromatin. The term ‘‘epigenetic’’ refers to the information contained in chromatin, other than the actual DNA sequence, that defines a heritable specific gene expression pattern. Epigenetically controlled genes are activated or repressed without any change in DNA. Perturbation of epigenetic balances may lead to alterations in gene expression, resulting in cellular transformation and malignant outgrowth. 1.2. Chromatin structure In all eukaryotes, DNA is compacted into the nucleus as chromatin. The traditional outlook is that chromatin is required for packing of the 1 m length of the human genome DNA into the 10 μm diameter average size human nucleus. However, our view on the function of chromatin has become broader and more dynamic than just that of a DNA-packaging device. Chromatin represents an additional level of regulation for all DNA metabolic processes (replication, repair and gene expression), working as a platform where biological signals integrate and molecular responses take 1 place. The structural unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around an octamer of very basic proteins called histones. Each nucleosome core consists of two copies of each of the histones: H2A, H2B, H3 and H4. These evolutionarily conserved proteins have a globular C-terminal domain critical to nucleosome formation and a flexible and unstructured N-terminal tail that protrudes from the nucleosome core. It remains unclear how nucleosomal arrays containing linker histone (H1) then twist and fold this chromatin fiber into increasingly more compacted filaments leading to defined higher order structures (Chodaparambil et al., 2006). Nucleosomes assemble on an 11 nm filament array known as beads on a string, which undergoes a series of wrapping and compacting events as cells progress from interphase to metaphase, culminating with the completely condensed chromosome during metaphase. Besides this generic organization, local chromosomal domains present different levels of structure: heterochromatin was originally identified cytogenetically as the portion of the genome that remains condensed after the transition from metaphase to interphase. These regions correspond to telomeres and pericentric chromosomal areas and generally localize attached to the perinuclear compartment. Heterochromatic areas tend to be rich in repetitive sequences, low in gene content (although some genes are present), transcriptionally silent or showing a variegating phenotype and typically replicate late. Euchromatin on the other hand may be considered as the rest of the genome, which de-condenses during interphase, contains most of the genes, is active or proficient for transcription and replicates early.